The process by which antibodies are formed is a complex one, many of the particulars of which are, however, understood. In brief, an involved set of reactions and responses are set in motion whenever a foreign material, such as an antigen, is introduced to an animal with a functioning immune system. Mature B cells are triggered to produce antibodies via their interaction with antigen and helper T cells. These antibody molecules consist of a light chain and a heavy chain, and are coded for by genes present in the mammalian genome. Every light chain is coded for by three distinct gene segments--the V.sub.L, J.sub.L and C.sub.L segments, while heavy chains are coded for by four segments, i.e., V.sub.H, D.sub.H, J.sub.H and C.sub.H.
In light chains, the variable region is coded for by the "V.sub.L " and "J.sub.L " segments, whereas the variable region of heavy chains is coded for by "V.sub.H ", "D.sub.H " and "J.sub.H " segments. A number of different genes exist for each segment. Shuffling and rearrangement can lead to an estimated 10.sup.6 -10.sup.7 antibodies, coded for by different combinations of variable gene segments. In addition, at all three points at which the variable gene segments are joined, i.e., the "V.sub.H -D", "D-J.sub.H " and "V.sub.L -J.sub.L " junctures, substantial sequence variability is possible for any given pair of assembling segments. This junctional variability, together with the combinational diversity, can lead to an estimated 10.sup.12 different antibodies. A composite variable region, formed by pairing of the heavy and light chain variable regions, contains the antibody's binding site. This binding site is of major interest in connection with the subject invention.
When an antibody response to a T cell dependent antigen is mounted, those B cells with antibodies capable of engaging the antigen proliferate to form large clones. In addition, members of the B cell clone diversify their variable genes by a hypermutation mechanism. This diversification of the B cell clone is of central important to the invention.
The somatic mutation process is well recognized in the art, but is poorly understood. See, e.g., Crews et al., Cell 25: 56 (1981); Gearhart et al., Nature 291: 29 (1981); Bothwell et al., Cell 24: 625 (1981); Siekevitz et al., Eur. J. Immunol. 13: 123 (1983); Clarke et al., J. Exp. Med. 161: 687 (1985); Selsing et al., Cell 25: 47 (1981).
The time period during which mutational diversification occurs is not known, although it is known that somatic mutations are acquired at some stage of the primary immune response, and possibly during secondary and later response (Griffith et al., J. Immunol. 312: 271 (1984); Wysocki et al., Proc. Natl. Acad. Sci. USA 83: 1847 (1986); Levy et al., J. Exp. Med. 169: 2007 (1989)). The mutation process introduces, e.g., point nucleotide substitutions in the assembled antibody genes expressed by clones of immune participating B lymphocytes. Often these nucleotide substitutions result in amino acid replacements in the encoded antibody variable region. In this way, the antibodies expressed by different members of a mutationally active B cell clone may differ in variable region sequence and potentially in binding site structure and function as well. Changes in the antibody binding site that are the direct consequences of the somatic mutation process are of central importance in connection with the subject invention.
Somatic mutations are almost always found in the variable genes of memory B cells when these are sampled by hybridoma production. It has also been observed that recruitment into the memory B cell compartment of the immune repertoire is strongly correlated with acquisition of specific somatic mutations and combinations thereof which confer upon antibody product increased affinity for immunizing antigen. (See Moller ed., "Role of somatic mutation in the generation of lymphocyte diversity" in Immunol. Rev. 96: 162 (1987)). These observations suggest that only a slender fraction of a mutationally diversified clone of B cells is usually recruited into the memory compartment. The unobserved majority of a diversified population must presumably include members with antibodies whose affinity for stimulatory antigen has been reduced or abolished. (See Manser et al., J. Exp. Med. 166: 1456 (1987)). Some fraction of this majority, however, presumably retains the ability to produce antibodies. It has now been found that these mutant B cells can be stimulated to proliferate into "subclones" which are specific to a chosen antigen which is distinguishable from the antigen used in the first immunization. These subclones result, surprisingly, via immunizing a subject animal with the antigen of choice following stimulation of the animal via, e.g., immunization with a first immunogen. In effect, one can recruit "mutant" B cells which bind to and are stimulated by an antigen of interest but are derived originally from precursor B cells which were incapable of being stimulated by the antigen of interest. The examples which follow set forth how this is accomplished.